Novel CACNA1C R511Q mutation, located in domain Ⅰ-Ⅱ linker, causes non-syndromic type-8 long QT syndrome

Background Gain-of-function mutations in CACNA1C encoding Cav1.2 cause syndromic or non-syndromic type-8 long QT syndrome (LQTS) (sLQT8 or nsLQT8). The cytoplasmic domain (D)Ⅰ-Ⅱ linker in Cav1.2 plays a pivotal role in calcium channel inactivation, and mutations in this site have been associated with sLQT8 (such as Timothy syndrome) but not nsLQT8. Objective Since we identified a novel CACNA1C mutation, located in the DⅠ-Ⅱ linker, associated with nsLQTS, we sought to reveal its biophysical defects. Methods Target panel sequencing was employed in 24 genotype-negative nsLQTS probands (after Sanger sequencing) and three family members. Wild-type (WT) or R511Q Cav1.2 was transiently expressed in tsA201 cells, then whole-cell Ca2+ or Ba2+ currents (ICa or IBa) were recorded using whole-cell patch-clamp techniques. Results We identified two CACNA1C mutations, a previously reported R858H mutation and a novel R511Q mutation located in the DⅠ-Ⅱ linker. Four members of one nsLQTS family harbored the CACNA1C R511Q mutation. The current density and steady-state activation were comparable to those of WT-ICa. However, persistent currents in R511Q-ICa were significantly larger than those of WT-ICa (WT at +20 mV: 3.3±0.3%, R511Q: 10.8±0.8%, P<0.01). The steady-state inactivation of R511Q-ICa was weak in comparison to that of WT-ICa at higher prepulse potentials, resulting in increased window currents in R511Q-ICa. Slow component of inactivation of R511Q-ICa was significantly delayed compared to that of WT-ICa (WT-tau at +20 mV: 81.3±3.3 ms, R511Q-tau: 125.1±5.0 ms, P<0.01). Inactivation of R511Q-IBa was still slower than that of WT-IBa, indicating that voltage-dependent inactivation (VDI) of R511Q-ICa was predominantly delayed. Conclusions Delayed VDI, increased persistent currents, and increased window currents of R511Q-ICa cause nsLQT8. Our data provide novel insights into the structure-function relationships of Cav1.2 and the pathophysiological roles of the DⅠ-Ⅱ linker in phenotypic manifestations.


Subjects and genetic analyses
This study was approved by Gunma University Ethical Review Board for Medical Research Involving Human Subjects (approval number: 2017-15). The subjects of this study were 24 genotype-negative nsLQTS probands remaining after screening major LQTS-related genes (KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2) using Sanger sequencing and three family members (II-3, III-2 and III-3) of one proband (III-1) (Fig 1A). Written informed consent for the genetic and functional analyses was obtained from the subjects. Clinical features of III-1, III-2 and II-3 and genetic information of major LQTS-related genes (KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2), obtained by Sanger sequencing, of III-1 and III-2 have already been reported [24].
A target panel sequencing of 72 genes, including LQTS-related genes, was employed in the subjects, as previously described [21]. Briefly, genomic DNA extracted from peripheral blood lymphocytes were subjected to custom panel sequencing using HaloPlex HS Custom (Agilent Technologies, Santa Clara, CA, USA) or xGen Predesigned Gene Capture Pools (Integrated DNA Technologies, Inc., Coralville, IA, USA) according to the manufacturer's protocol. The resulting library was subjected to the paired-end sequencing of 150 bp reads on MiSeq (Illumina, San Diego, CA, USA). A bioinformatics analysis for HaloPlex HS Custom data was performed as previously described [21]. Reads obtained from xGen Predesigned Gene Capture Pools were subsequently aligned against hg19 using BWA and variant calling was performed using GATK in the BWA Enrichment v2.1.2 application on the BaseSpace Sequence Hub (Illumina) with default settings. The mean region coverage depth of 27 samples was 243 (150-426), and the percentage with target coverage of at least 20X was 98.8 (97.8-100). Among them, the mean region coverage depth was 327.9 (a CACNA1C R858H carrier), 196.4 (II-3), 204.8 (III-1), 290.1 (III-2) and 274 (III-3) (Fig 1A), and the percentage with target coverage of at least 20X was ≧99.9 in these cases. The identified variants, c.2573G>A/p.R858H in exon 19 and c.1532G>A/p.R511Q in exon 12 of CACNA1C (NM_000719.7), were validated by Sanger sequencing.

Electrophysiology
Membrane calcium or barium currents (I Ca or I Ba ) were recorded using whole-cell patchclamp techniques at room temperature (23-25˚C). Since electrophysiological studies of most gain-of-function CACNA1C mutations in the DI-II linker have been conducted under the condition of high extracellular calcium concentration, we also used a bath solution with a high calcium concentration. The bath solution contained (in mmol/L) 130 N-methyl-d-glucamine, 5 KCl, 15 CaCl 2 (for I Ca ) or BaCl 2 (for I Ba ), 1 MgCl 2 and 10 HEPES (pH 7.35 with HCl), and the pipette solution contained (in mmol/L) 120 CsCl, 2 MgCl 2 , 2 MgATP, 5 CaCl 2 , 10 EGTA and 10 HEPES (pH 7.25 with CsOH). The electrode resistance ranged from 1.5 to 2.0 MO. Data acquisition was carried out using an Axopatch 200B amplifier and pCLAMP10.3 software (Molecular Devices, Sunnyvale, CA, USA). Currents were acquired at 20-50 kHz, and low pass-filtered at 5 kHz using an analog-to-digital interface (Digidata 1440A acquisition system, Molecular Devices). Current densities at each test potential were obtained by dividing the calcium currents by cell capacitance. The steady-state activation and steady-state inactivation curves were fitted with Boltzmann functions of the following forms: A 0 +A f [1-exp(-t/τf)]+A s [1-exp(-t/τs)], where A and τ refer to the amplitudes and time constants, respectively, and f and s refer to the fast and slow components, respectively. I refers to the current, and t refers to the time. The time course of recovery from inactivation was fitted with a single exponential function of the following form: I(t)/I max = A 0 +A 1 exp(-t/τ), as described previously [25,26]. To avoid potential endogenous current contamination, recordings from the cells exhibiting peak inward current amplitudes of <0.3 nA were excluded from the analyses of the inactivation rate, steady-state inactivation and time courses of inactivation and recovery from inactivation.

Statistical analysis
All data are expressed as mean ± standard error, and statistical comparisons were tested using the unpaired Student's t-test with p<0.05 considered to be statistically significant. In some figures, the standard error bars are smaller than the data symbols.

Identification of two CACNA1C mutations
We performed target panel sequencing in 24 genotype-negative nsLQTS probands after Sanger sequencing. As a result, we identified two CACNA1C mutations, an R858H mutation, which has already been reported, and a novel R511Q mutation, but did not detect any other pathogenic variants in LQTS-related genes [19]. Since the CACNA1C R858H mutation has already been functionally characterized by Fukuyama et al., we report the clinical characteristics of carriers of the novel CACNA1C R511Q mutation and its biophysical defects [19].

Case presentations harboring the CACNA1C R511Q mutation
The index patient (III-3) (Fig 1A), a 17-year-old female at the time of the genetic test, had been asymptomatic with QT prolongation that had first been identified at 7 years of age. She was a younger paternal half-sister of the deceased case (III-1) (Fig 1A) we had previously reported [24]. Her ECG showed sinus rhythm with QT prolongation (HR: 98 bpm, QTc: 506 ms) ( Fig  1B). An echocardiogram revealed no structural heart disease. Neither she nor any other family members had any cardiac or extra-cardiac abnormalities, except for QT prolongation. We first examined whether or not the index patient (III-3) (Fig 1A) carried the KCNE1 D85N variant, as with the proband (III-1) (Fig 1A), using Sanger sequencing. However, we did not detect it, suggesting that other genetic factor(s) might be associated with this nsLQTS family.
ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/190641/) indicated that the variant was uncertain significance (Table 1). On the other hand, the KCNE1 D85N variant was found in III-1 and III-2, as reported previously, but not in III-3 and II-3 (Table 1) (Fig 1A), suggesting that the CACNA1C R511Q variant might play a more important role in the generation of phenotypes than the KCNE1 D85N variant [24].

Biophysical defects of the CACNA1C R511Q mutation
To examine the pathogenicity of the CACNA1C R511Q variant, we conducted a functional analysis by measuring whole-cell membrane currents using Ca 2+ as a charge carrier (I Ca ). As shown in Fig 2A, WT CACNA1C exhibited I Ca (WT-I Ca ), and R511Q CACNA1C exhibited I Ca (R511Q-I Ca ) resembling WT-I Ca . The peak current density, measured at +20 mV from a holding potential of -70 mV, and steady-state activation of R511Q-I Ca were comparable to those of WT-I Ca ( Table 2) (Fig 2B and 2C).
However, residual currents at 100-ms (r 100 ) of depolarization potentials (+20 mV, +30 mV and +40 mV) in R511Q-I Ca were significantly larger than those in WT-I Ca (Table 3) ( Fig 2D). Notably, although r 100 s of WT-I Ca decreased with higher depolarization potentials, those of R511Q-I Ca were maintained. On the other hand, residual persistent currents at 300-ms (r 300 ) of depolarizing potentials (+10 mV, +20 mV and +30 mV) in R511Q-I Ca were significantly larger than those in WT-I Ca (Table 3) (Fig 2D). Although r 300 s of WT-I Ca markedly decreased with higher depolarization potentials, those of R511Q-I Ca showed a mild decrease. Thus, the difference in residual currents between WT-I Ca and R511Q-I Ca became larger with higher depolarization potentials. I Ca displays two forms of inactivation: voltage-dependent inactivation (VDI) and calciumdependent inactivation (CDI) [3,27,28]. CDI and VDI are thought to correspond to the fast and slow component of inactivation, respectively. Inactivating currents of WT-I Ca and R511Q-I Ca during 300-ms depolarizing potentials from +10 mV to +40 mV could be fitted by a double exponential function. The time constants of fast components of R511Q-I Ca were comparable to those of WT-I Ca at each test potential, although the amplitudes of fast components of R511Q-I Ca became smaller than those of WT-I Ca as the depolarizing potentials became higher (Table 3) (Fig 2E). In contrast, the time constants of slow components of R511Q-I Ca were significantly larger than those of WT-I Ca at each test potential, and the amplitudes of slow component of R511Q-I Ca became larger than those of WT-I Ca as the depolarizing potentials became higher (Table 3) (Fig 2E). These findings suggest that, in R511Q-I Ca , the CDI decreased and VDI increased with slower inactivation, especially at higher depolarization potentials. The steady-state inactivation was assessed using a pulse protocol shown in the inset in Fig  2C. The voltage at which half of the channels are available to open (V 1/2 ) of R511Q-I Ca was comparable to that of WT-I Ca (Table 2) (Fig 2C). However, the slope factor (K) of R511Q-I Ca was significantly larger than that of WT-I Ca ( Table 2) (Fig 2C), and the I/I max of R511Q-I Ca  was significantly larger than that of WT-I Ca when prepulse potentials were between 0 mV and +50 mV (Fig 2C), possibly due to larger persistent currents during prepulse potentials in R511Q-I Ca , which resulted in increased window currents in R511Q-I Ca in comparison to those in WT-I Ca (Fig 2C). The recovery from inactivation was assessed using a double pulse protocol shown in the inset in Fig 2F, and plots were fitted by a single exponential function (Fig 2F). Although the P2/P1 of R511Q-I Ca was significantly larger than that of WT-I Ca from the beginning of an interpulse duration of several ms, possibly due to larger persistent currents in R511Q-I Ca during P1 pulses, the time constant of R511Q-I Ca was comparable to that of WT-I Ca (WT-tau: 135 ±3.7 ms, n = 7, R511Q-tau: 145±7.6 ms, n = 9, P = NS). This indicated that the time course of recovery from inactivation of R511Q-I Ca was not markedly different from that of WT-I Ca .
The VDI can be studied with Ba 2+ as the charge carrier, which excludes the CDI process [28]. Therefore, the Ca 2+ in the bath solution was replaced with Ba 2+ , and then Ba 2+ currents through calcium channels (I Ba ) were recorded. As shown in Fig 3A, WT-I Ba and R511Q-I Ba exhibited delayed inactivation compared to WT-I Ca and R511Q-I Ca , respectively. Inactivating currents of WT-I Ba and R511Q-I Ba during depolarizing potentials from +10 mV to +40 mV could be fitted by a single exponential function. The time constants of R511Q-I Ba were still larger than those of WT-I Ba at each test potential (Fig 3B), suggesting that the VDI rather than the CDI of R511Q might be impaired. The VDI was presented as the fraction of current remaining after a 300-ms depolarization normalized to peak (r 300 ) across various voltages, and the extent of CDI was calculated as f 300 = (r 300Ba -r 300Ca )/r 300Ba [18]. The VDI of R511Q-I Ba and R511Q-I Ca was significantly delayed compared to that of WT-I Ba and WT-I Ca , respectively ( Fig 3C). However, the f 300 for WT at +20 mV (WT-f 300 ) and f 300 for R511Q at +20 mV (R511Q-f 300 ) were 0.67±0.05 (n = 5) and 0.67±0.05 (n = 4), respectively (p = NS). Taken together, these results indicated that the VDI of R511Q was predominantly impaired compared to the CDI.

Identification of two CACNA1C mutations associated with nsLQT8
We identified two CACNA1C mutations, using target panel sequencing, in 24 genotype-negative nsLQTS patients after screening of major LQTS-related genes using Sanger sequencing. This appears to be consistent with the notion that nsLQT8 is more prevalent than previously expected [19,20,29,30]. Therefore, the CACNA1C should be involved in genes for screening LQTS patients.
Gain-of-function of CACNA1C mutations associated with QT prolongation can be caused by multiple mechanisms, including delayed inactivation, increased persistent currents, increased window currents, increased current density, a combination thereof and increased permeability of non-selective monovalent cations [14]. However, the mechanisms that cause other cardiac and extra-cardiac phenotypes remain unknown.

Novel CACNA1C R511Q mutation and its biophysical defects
We identified the CACNA1C R511Q mutation, located in the DI-II linker, in four patients in one LQTS family. All patients harboring the mutation exhibited QT prolongation but no other cardiac or extra-cardiac phenotypes.
A functional study using a heterologous expression system revealed that the CACNA1C R511Q displayed a delay of the slow component of I Ca inactivation, increased persistent currents, and increased window currents, without a change of the current density.
The inactivation of I Ca occurs via two mechanisms: the VDI, which is linked to the change of transmembrane potential, and the CDI, which is mediated by calcium ions that carry the current [3,27,28]. The molecular determinants of the VDI include the cytoplasmic ends of the S6 segments, DI-II linker, and C-terminus of Cav1.2 [22]. In particular, the DI-II linker has been suggested to be a particle that occludes the channel pore during inactivation [22,23]. In contrast, the molecular determinants of the CDI include C-terminus [22,28,31,32].
When Ba 2+ is used as a charge carrier (I Ba ), the fast component of inactivation is lost and the slow component of inactivation is markedly decelerated [28]. In our experiments, inactivating Ba 2+ currents in both WT-I Ba and R511Q-I Ba could be fitted by a single exponential function rather than a double exponential function, possibly due to a loss of the fast component of I Ca inactivation. Time constants of inactivating currents in R511Q-I Ba were still larger than those in WT-I Ba , indicating that the VDI of R511Q-I Ca was predominantly decelerated compared to that of WT-I Ca . Furthermore, the extent of calculated CDI was not different between WT and R511Q, which also indicated that the VDI of R511Q-I Ca was predominantly decelerated.

CACNA1C mutations in the DIS6 or DI-II linker
Focusing on mutations in the DIS6 and DI-II linker, those for TS1 and TS2 (G406R-8A, G402S-8 and G406R-8) displayed a marked delay of I Ca inactivation, marked increases of persistent currents and window currents (Table 4) [7,8]. A G419R mutation for aTS displayed an increased current density with accelerated inactivation (Table 4) [17]. Mutations, R518H and R518C for COTS, displayed a delay of I Ca inactivation, increased persistent currents and increased window currents (but those were weaker than TS mutations: G406R-8A, G402S-8 and G406R-8) with a reduced current density (Table 4) [18]. In our study, the R511Q mutation displayed a delay of I Ca inactivation, increased persistent currents, which is very likely the main actor of QT prolongation, and increased window currents (but those were weaker than TS mutations: G406R-8A, G402S-8 and G406R-8) without a change of current density. These biophysical defects of the R511Q mutation were more modest than seen in other mutations for TS, aTS or COTS (Table 4), which may be the reason why the R511Q mutation is associated with nsLQT8 but not with other cardiac and extra-cardiac phenotypes. Otherwise, TS mutations have been reported to impair not only the VDI but also the CDI, suggesting that an impairment of the CDI may be associated with the manifestation of other cardiac or extra-cardiac phenotypes [7,[33][34][35]. These findings provide a novel insight into the pathophysiological roles of the DI-II linker in phenotypic manifestations. Korkosh et al. recently built structural models of Cav1.2 and proposed mechanisms underlying the VDI: The cytoplasmic N-terminal part of VSDII (DIIS0) in the DI-II linker is bound with the α1-interaction domain (AID). Following voltage-dependent channel activation, the cytoplasmic face of DIIS0 would perturb and shift DIIS0-bound AID toward the pore axis. The AID-linked DIS6 would bend at the flexible G402 and G406, facilitating the activationgate closure and thus the VDI [36]. The R511 position in DIIS0 may form a salt bridge with acidic residues in positions immediately C-terminal to the AID. Therefore, the R511Q mutation would destroy the salt bridges and weaken the DIIS0-AID contact, retarding the AID displacement thus delaying the VDI. Regarding the interaction of AID and β-subunit, four basic residues (R514, R515, R518 and K522) in DIIS0 are thought to provide large contributions to the interaction energy between AID and β-subunit, while R511 is not [36]. Further studies are required to reveal whether or not R511Q affects the DIIS0-AID-β-subunit interaction.

Other genetic variants in this nsLQTS family
All four patients presenting with QT prolongation in the family carried the CACNA1C R511Q mutation, but not all carried the KCNE1 D85N variant which was reported to be a diseasecausing or a drug-induced LQTS variant [24,39,40]. This suggested that the CACNA1C R511Q mutation might be a main contributor to QT prolongation in this family, and the KCNE1 D85N variant may have contributed less to QT prolongation in comparison to the CACNA1C R511Q mutation. Although the father (II-3) (Fig 1A) harbors the CACNA1C R511Q mutation, his QTc interval was only slightly prolonged. This may be due to the fact that the QTc interval in males is shorter than that in females. The contribution of these variants to QT prolongation and cardiac events in this family needs to be further investigated.

Conclusions
Target panel sequencing in 24 genotype-negative nsLQTS patients after Sanger screening identified two CACNA1C mutations: An R858H mutation in one proband and a novel R511Q mutation in one family. This supported the notion that nsLQT8 is more prevalent than previously expected. Despite the fact that CACNA1C mutations in the DI-II linker have been associated with sLQTS (TS, aTS and COTS), the R511Q mutation in this site is associated with nsLQT8. The biophysical defects of the R511Q mutation were a delay of slow component of I Ca inactivation through predominant impairment of the VDI, increased persistent currents, and increased window currents, without a change of the current density. The degree of functional impairment associated with the R511Q was more modest in comparison to that seen with TS, aTS and COTS mutations. These findings provide novel insights into the structurefunction relationships of Cav1.2 and the pathophysiological roles of the DI-II linker in phenotypic manifestations.